Heterocyclic substituted 1,4-dihydro-4-oxo-1,8-naphthpyridine analogs

ABSTRACT

The present invention relates to 1,4-dihydro-4-oxo-1,8-napthpyridine analogs of the formula  
                 
and pharmaceutically acceptable salts, esters, and prodrugs thereof, wherein A, X, W and Y are substituents. The present invention also relates to methods for using such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. provisional application60/461,205, filed Apr. 7, 2003, and 60/519,569, filed Nov. 12, 2003. Thecontents of these documents are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to substituted 1,4-dihydro-4-oxo-1,8-napthpyridineanalogs, and treatment methods using such compounds.

BACKGROUND

Quadruplexes can form in certain purine-rich strands of nucleic acids.In duplex nucleic acids, certain purine rich strands are capable ofengaging in a slow equilibrium between a typical duplex helix structureand in unwound and non-B-form regions. These unwound and non-B forms canbe referred to as “paranemic structures.” Some forms are associated withsensitivity to S1 nuclease digestion, which can be referred to as“nuclease hypersensitivity elements” or “NHEs.” A quadruplex is one typeof paranemic structure and certain NHEs can adopt a quadruplexstructure. Considerable circumstantial evidence suggests that quadruplexstructures can exist in vivo in specific regions of the genome,including the telomeric ends of chromosomes and oncogene regulatoryregions. (Han, et al., Trends Pharm. Sci. (2000) 21:136-142). Thus,quadruplex forming regions of DNA may be used as molecular targets foranticancer agents.

SUMMARY OF THE INVENTION

Compounds described herein interact with regions of DNA that can formquadruplexes and act as tumor suppression genes with high affinity. Suchcompounds can reduce expression of highly proliferate genes and areutilized for treating cancers. Furthermore, the compounds may alsoexhibit antibacterial or antiviral activity, and may be used fortreating bacterial and viral infections.

Various embodiments of the compounds of the present invention aredescribed below. The present invention encompasses other compoundshaving formula 1, with substituents independently selected fromcompounds in Table 1. Thus, the present invention is not limited to thespecific combination of substituents described in various embodimentsbelow.

The compounds have the general formula:

and pharmaceutically acceptable salts, esters and prodrugs thereof;

wherein W and Z are independently OR² or NR¹R² wherein R¹ and R² mayform an optionally substituted ring;

A is H, halo or NR¹ ₂;

R¹ is H or a C₁₋₆ alkyl;

R² is H or a C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl optionally containing one ormore non-adjacent heteroatoms selected from N, O, and S, and optionallysubstituted with a carbocyclic or heterocyclic ring; or R¹ is anoptionally substituted heterocyclic ring, aryl or heteroaryl;

Y is selected from the group consisting of

where R⁶ is a substituent at any position on the fused ring; and is H,OR¹, C₁₋₆ alkyl, C₂₋₆ alkenyl, each optionally substituted by halo, ═Oor one or more heteroatoms; or R⁵ is an inorganic substituent; or twoadjacent R⁶ is linked to obtain a 5-6 membered substituted orunsubstituted carbocyclic or heterocyclic ring, optionally fused to anadditional substituted or unsubstituted carbocyclic or heterocyclicring;

-   -   Q is CH or N;    -   and X is O, NH, or S;

provided that W is not hydroxy or ethoxy when Y is 2-thiazolyl or Z is3-amino-1-pyrrolidinyl.

In the above formula 1, A may be halo. In one example, A is fluoro.

In the above formula 1, Y may have the formula

where X is S and R₆ is H;

or the formula

where X is S, Q is CH, and R₆ is H.

In the above formula 1, W and Z may independently be NR¹R². In oneexample, R¹ is H and R² is a C₁₋₁₀ alkyl optionally containing one ormore heteroatoms, and optionally substituted with a C₃₋₆ cycloalkyl,aryl or a 5-14 membered heterocyclic ring containing one or more N, O orS. In another example, R¹ is H and R² is an aryl or a 5-14 memberedheterocyclic ring containing one or more N, O or S, each optionallysubstituted with an amino or another heterocyclic ring. In yet anotherexample, R¹ and R² in NR¹R² form an optionally substituted 5-14 memberedring containing one or more N, O or S. In particular examples, NR¹R² ismorpholine, thiomorpholine, piperazine, piperidine or diazepine.

In the above formula 1, W and Z may independently have the formulaNR¹—(CR¹ ₂)n-NR³R⁴  (2)

wherein R¹ and R³ are independently H or C₁₋₆ alkyl;

n is 1-6; and

R⁴ is H or a C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl optionally containing one ormore non-adjacent heteroatoms selected from N, O and S, and optionallysubstituted with a carbocyclic or heterocyclic ring; and

wherein in NR³R⁴, R³ and R⁴ may form an optionally substituted ring.

In the above formula 2, n may be 2-3. In one example, NR³R⁴ is anacyclic amine, or guanidinyl or a tautomer thereof; or R³ and R⁴optionally form a substituted ring containing one or more N, O or S. Inparticular examples, NR³R⁴ is morpholine, thiomorpholine, imidazole,pyrrolidine, piperazine, pyridine or piperidine.

In the above formula 1, Z may be NR¹R²; and W may have the formulaNR¹—(CR¹ ₂)_(n)—NR³R⁴  (2)

wherein R¹ and R² are as defined in claim 1;

R³ is H or C₁₋₆ alkyl;

n is 1-6; and

R⁴ is H or a C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl optionally containing one ormore non-adjacent heteroatoms selected from N, O and S, and optionallysubstituted with a carbocyclic or heterocyclic ring; and

wherein in NR¹R² and NR³R⁴, R¹ and R², and R³ and R⁴ each independentlymay form a substituted ring.

In the above formula 2, where Z is NR¹R²; and W has the formula NR¹—(CR¹₂)_(n)—NR³R⁴ (2), R¹ and R² in NR¹R², and R³ and R⁴ in NR³R⁴ each mayindependently form a substituted ring containing one or more N, O or S.For example, Z is optionally substituted with amino, carbamate, a C₁₋₁₀alkyl containing one or more non-adjacent N, O or S, and optionallysubstituted with a heterocyclic ring; aryl or a saturated or unsaturatedheterocyclic ring, each of which is optionally substituted. In oneexample, Z and NR³R⁴ are independently morpholine, thiomorpholine,imidazole, pyrrolidine, piperazine, pyridine or piperidine. In oneexample, Z and NR³R⁴ are independently pyrrolidine. In another example,Z is pyrrolidine substituted with pyrazine.

Examples of 5-6 membered heterocyclic rings include but are not limitedto tetrahydrofuran, 1,3-dioxolane, 2,3-dihydrofuran, tetrahydropyran,benzofuran, isobenzofuran, 1,3-dihydro-isobenzofuran, isoxazole,4,5-dihydroisoxazole, piperidine, pyrrolidine, pyrrolidin-2-one,pyrrole, pyridine, pyrimidine, octahydro-pyrrolo[3,4-b]pyridine,piperazine, pyrazine, morpholine, thiomorpholine, imidazole,imidazolidine-2,4-dione, benzimidazole, 1,3-dihydrobenzimidazol-2-one,indole, thiazole, benzothiazole, thiadiazole, thiophene,tetrahydro-thiophene 1,1-dioxide, diazepine, triazole, guanidine,diazabicyclo[2.2.1]heptane, 2,5-diazabicyclo[2.2.1]heptane, and2,3,4,4a,9,9a-hexahydro-1H-β-carboline.

In the above formula 1, Z may be OR² and R² is a C₁₋₆ alkyl optionallysubstituted with a carbocyclic or heterocyclic ring.

In the above formula 1, each optionally substituted moiety issubstituted with one or more halo, OR², NR¹R², carbamate, C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, each optionally substituted by halo, ═O, aryl or one ormore heteroatoms; inorganic substituents, aryl, carbocyclic or aheterocyclic ring.

The compounds of the present invention may be chiral. As used herein, achiral compound is a compound that is different from its mirror image,and has an enantiomer. Methods of synthesizing chiral compounds andresolving a racemic mixture of enantiomers are well known to thoseskilled in the art. See, e.g., March, “Advanced Organic Chemistry,” JohnWiley and Sons, Inc., New York, (1985), which is incorporated herein byreference.

The present invention also provides pharmaceutical compositionscomprising compounds having formula 1, and a pharmaceutically acceptableexcipient.

The present invention also provides methods for identifying a compoundthat interacts with a quadruplex-forming region of DNA, comprising

a) contacting a nucleic acid capable of forming a quadruplex with aprimer comprising a label to form a complex;

b) contacting said complex with one or more test compounds and apolymerase to form a reaction mixture, and

c) separating said reaction mixture by capillary electrophoresis toobtain one or more reaction products; and

d) determining the extent of primer extension in said one or morereaction products.

In one example, the method further comprises the step of determining thebinding affinity of said one or more test compounds for said nucleicacid. In a particular example, the label is a fluorescent label.

Furthermore, the present invention provides methods for ameliorating acell proliferative disorder, comprising administering to a subject inneed thereof an effective amount of a compound having formula 1 or apharmaceutical composition thereof, thereby ameliorating saidcell-proliferative disorder. In one example, the cell proliferativedisorder is cancer. In another example, cell proliferation is reduced,or cell death is induced. The subject may be human or animal.

The present invention also provides methods for reducing cellproliferation or inducing cell death, comprising contacting a systemwith an effective amount of a compound having formula 1 or apharmaceutical composition thereof, thereby reducing cell proliferationor inducing cell death in said system. The system may be a cell ortissue.

The present invention further provides methods for reducing microbialtiters, comprising contacting a system with an effective amount of acompound having formula 1 or a pharmaceutical composition thereof,thereby reducing microbial titers. The system may be a cell or tissue.In one example, the microbial titers are viral, bacterial or fungaltiters.

Further, the present invention provides methods for ameliorating amicrobial infection, comprising administering to a subject in needthereof an effective amount of a compound having formula 1 or apharmaceutical composition thereof, thereby ameliorating said microbialinfection. The subject may be human or animal. In one example, themicrobial infection is viral, bacterial or fungal.

DESCRIPTION OF THE INVENTION

The present invention relates to 1,4-dihydro-4-oxo-1,8-naphthpyridineanalogs having formula I,

and pharmaceutically acceptable salts, esters, and prodrugs thereof.

In particular embodiments, the compounds interact with regions of DNAthat can form quadruplexes. The present invention also relates tomethods for treating cancer, bacterial and viral infections using suchcompounds.

Because regions of DNA that can form quadruplexes are regulators ofbiological processes such as oncogene transcription, modulators ofquadruplex biological activity can be utilized as cancer therapeutics.Molecules that interact with regions of DNA that can form quadruplexescan exert a therapeutic effect on certain cell proliferative disordersand related conditions. Particularly, abnormally increased oncogeneexpression can cause cell proliferative disorders and quadruplexstructures typically down-regulate oncogene expression. Examples ofoncogenes include but are not limited to MYC, HIF, VEGF, ABL, TGF,PDGFA, MYB, SPARC, HUMTEL, HER, VAV, RET, H-RAS, EGF, SRC, BCL1, BCL2,and other oncogenes known to one of skill in the art.

Molecules that bind to regions of DNA that can form quadruplexes canexert a biological effect according to different mechanisms, whichinclude for example, stabilizing a native quadruplex structure,inhibiting conversion of a native quadruplex to duplex DNA by blockingstrand cleavage, and stabilizing a native quadruplex structure having aquadruplex-destabilizing nucleotide substitution and other sequencespecific interactions. Thus, compounds that bind to regions of DNA thatcan form quadruplexes described herein may be administered to cells,tissues, or organisms for the purpose of down-regulating oncogenetranscription and thereby treating cell proliferative disorders. Theterms “treatment” and “therapeutic effect” as used herein refer toreducing or stopping a cell proliferation rate (e.g., slowing or haltingtumor growth) or reducing the number of proliferating cancer cells(e.g., removing part or all of a tumor).

Determining whether the biological activity of native DNA that can formquadruplexes is modulated in a cell, tissue, or organism can beaccomplished by monitoring quadruplex biological activity. Quadruplexforming regions of DNA biological activity may be monitored in cells,tissues, or organisms, for example, by detecting a decrease or increaseof gene transcription in response to contacting the quadruplex formingDNA with a molecule. Transcription can be detected by directly observingRNA transcripts or observing polypeptides translated by transcripts,which are methods well known in the art.

Molecules that interact with quadruplex forming DNA and quadruplexforming nucleic acids can be utilized to treat many cell proliferativedisorders. Cell proliferative disorders include, for example, colorectalcancers and hematopoietic neoplastic disorders (i.e., diseases involvinghyperplastic/neoplastic cells of hematopoietic origin such as thosearising from myeloid, lymphoid or erythroid lineages, or precursor cellsthereof). The diseases can arise from poorly differentiated acuteleukemias, e.g., erythroblastic leukemia and acute megakaryoblasticleukemia. Additional myeloid disorders include, but are not limited to,acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) andchronic myelogenous leukemia (CML) (Vaickus, Crit. Rev. inOncol./Hemotol. 11:267-297 (1991)). Lymphoid malignancies include, butare not limited to acute lymphoblastic leukemia (ALL), which includesB-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease. Cell proliferative disorders also includecancers of the colorectum, breast, lung, liver, pancreas, lymph node,colon, prostate, brain, head and neck, skin, liver, kidney, and heart.Compounds that interact with regions of DNA that can form quadruplexesalso can be utilized to target cancer related processes and conditions,such as increased angiogenesis, by inhibiting angiogenesis in a subject.

The present invention provides a method for reducing cell proliferationor for treating or alleviating cell proliferative disorders, comprisingcontacting a system having a native DNA capable of forming a quadruplexregion with a compound having formula I. The system may be a group ofcells or one or more tissues. In one embodiment, the system is a subjectin need of a treatment of a cell proliferative disorder (e.g., a mammalsuch as a mouse, rat, monkey, or human). The present invention alsoprovides a method for treating colorectal cancer by administering acompound that interacts with a c-MYC quadruplex forming region to asubject in need thereof, thereby reducing the colorectal cancer cellproliferation. Furthermore, the present invention provides a method forinhibiting angiogenesis and optionally treating a cancer associated withangiogenesis, comprising administering a compound that interacts with avascular endothelial growth factor (VEGF) quadruplex forming region to asubject in need thereof, thereby reducing angiogenesis and optionallytreating a cancer associated with angiogenesis.

As used herein, the terms “treat,” “treatment” and “therapeutic effect”refer to reducing or stopping a cell proliferation rate (e.g., slowingor halting tumor growth) or reducing the number of proliferating cancercells (e.g., removing part or all of a tumor). These terms also areapplicable to reducing a titre of a microorganism in a system (i.e.,cell, tissue, or subject) infected with a microorganism, reducing therate of microbial propagation, reducing the number of symptoms or aneffect of a symptom associated with the microbial infection, and/orremoving detectable amounts of the microbe from the system. Examples ofmicroorganism include but are not limited to virus, bacterium andfungus.

Compounds that interact with quadruplex forming regions of DNA can alsobe used to reduce a microbial infection, such as a viral infection.Retroviruses offer a wealth of potential targets for G-quadruplextargeted therapeutics. G-quadruplex structures have been implicated asfunctional elements in at least two secondary structures formed byeither viral RNA or DNA in HIV, the dimer linker structure (DLS) and thecentral DNA flap (CDF). Additionally, DNA aptamers which are able toadopt either inter- or intramolecular quadruplex structures are able toinhibit viral replication. In one example, DNA aptamers are able toinhibit viral replication by targeting the envelope glycoprotein(putatively). In another example, DNA aptamers inhibit viral replicationby targeting the HIV-integrase respectively, suggesting the involvementof native quadruplex structures in interaction with the integraseenzyme.

Dimer linket structures, which are common to all retroviruses, serve tobind two copies of the viral genome together by a non-covalentinteraction between the two 5′ ends of the two viral RNA sequences. Thegenomic dimer is stably associated with the gag protein in the maturevirus particle. In the case of HIV, the origin of this non-covalentbinding may be traced to a 98 base-pair sequence containing several runsof at least two consecutive guanines (e.g., the 3′ for the formation ofRNA dimers in vitro). An observed cation (potassium) dependence for theformation and stability of the dimer in vitro, in addition to thefailure of an antisense sequence to effectively dimerize, has revealedthe most likely binding structure to be an intermolecular G-quadruplex.

Prior to integration into the host genome, reverse transcribed viral DNAforms a pre-integration complex (PIC) with at least two major viralproteins, integrase and reverse transcriptase, which is subsequentlytransported into the nucleus. The Central DNA Flap (CDF) refers to99-base length single-stranded tail of the +strand, occurring near thecenter of the viral duplex DNA, which is known to a play a role in thenuclear import of the PIC. Oligonucleotide mimics of the CDF have beenshown to form intermolecular G-quadruplex structures in cell-freesystems.

Thus, compounds that recognize quadruplex forming regions can be used tostabilize the dimer linker structure and thus prevent de-coupling of thetwo RNA strands. Also, by binding to the quadruplex structure formed bythe CDF, protein recognition and/or binding events for nuclear transportof the PIC may be disrupted. In either case, a substantial advantage canexist over other anti-viral therapeutics. Current Highly ActiveAnti-Retroviral Therapeutic (HAART) regimes rely on the use ofcombinations of drugs targeted towards the HIV protease and HIVintegrase. The requirement for multi-drug regimes is to minimize theemergence of resistance, which will usually develop rapidly when agentsare used in isolation. The source of such rapid resistance is theinfidelity of the reverse transcriptase enzyme which makes a mutationapproximately once in every 10,000 base pairs. An advantage of targetingviral quadruplex structures over protein targets, is that thedevelopment of resistance is slow or is impossible. A point mutation ofthe target quadruplex can compromise the integrity of the quadruplexstructure and lead to a non-functional copy of the virus. A singletherapeutic agent based on this concept may replace the multiple drugregimes currently employed, with the concomitant benefits of reducedcosts and the elimination of harmful drug/drug interactions.

The present invention provides a method for reducing a microbial titerin a system, comprising contacting a system having a native DNAquadruplex forming region with a compound having formula I. The systemmay be one or more cells or tissues. Examples of microbial titersinclude but are not limited to viral, bacterial or fungal titers. In aparticular embodiment, the system is a subject in need of a treatmentfor a viral infection (e.g., a mammal such as a mouse, rat, monkey, orhuman). Examples of viral infections include infections by a hepatitisvirus (e.g., hepatitis B or C), human immunodeficiency virus (HIV),rhinovirus, herpes-zoster virus (VZV), herpes simplex virus (e.g., HSV-1or HSV-2), cytomegalovirus (CMV), vaccinia virus, influenza virus,encephalitis virus, hantavirus, arbovirus, West Nile virus, humanpapilloma virus (HPV), Epstein-Barr virus, and respiratory syncytialvirus. The present invention also provides a method for treating HIVinfection by administering a compound having formula I to a subject inneed thereof, thereby reducing the HIV infection.

Identifying Compounds that can Bind to Quadruplex Forming Regions of DNA

Compounds described herein are identified as compounds that can bind toquadruplex forming regions of DNA where a biological activity of thisregion, often expressed as a “signal,” produced in a system containingthe compound is different than the signal produced in a system notcontaining the compound. While background signals may be assessed eachtime a new molecule is probed by the assay, detecting the backgroundsignal is not required each time a new molecule is assayed.

Examples of quadruplex forming nucleotide sequences are set forth in thefollowing Table A: SEQ SEQUENCE ID NO ORIGIN TG₄AG₃TG₄AG₃TG₄AAGG 1 CMYCGGGGGGGGGGGGGCGGGGGCGGGGGCGGGGGAGGGGC 2 PDGFAG₈ACGCG₃AGCTG₅AG₃CTTG₄CCAG₃CG₄CGCTTAG₅ 3 PDGFB/c- sisAGGAAGGGGAGGGCCGGGGGGAGGTGGC 4 CABL AGGGGCGGGGCGGGGCGGGGGC 5 RETGGGAGGAAGGGGGCGGGAGCGGGGC 6 BCL-2 GGGGGGCGGGGGCGGGCGCAGGGGGAGGGGGC 7Cyclin D1/BCL-1 CGGGGCGGGGCGGGGGCGGGGGC 8 H-RASAGAGGAGGAGGAGGTCACGGAGGAGGAGGAGAAGGAGGAGGAGGAA 9 CMYB (GGA)₄ 10 VAVAGAGAAGAGGGGAGGAGGAGGAGGAGAGGAGGAGGCGC 11 HMGA2 GGAGGGGGAGGGG 12 CPIMAGGAGAAGGAGGAGGTGGAGGAGGAGG 13 HER2/neuAGGAGGAGGAGAATGCGAGGAGGAGGGAGGAGA 14 EGFRGGGGCGGGCCGGGGGCGGGGTCCCGGCGGGGCGGAG 15 VEGF CGGGAGGAGGAGGAAGGAGGAAGCGCG16 CSRC

In addition to determining whether a test molecule or test nucleic acidgives rise to a different signal, the affinity of the interactionbetween the nucleic acid and the compound may be quantified. IC₅₀,K_(d), or K_(i) threshold values may be compared to the measured IC₅₀ orK_(d) values for each interaction, and thereby identify a test moleculeas a quadruplex interacting molecule or a test nucleic acid as aquadruplex forming nucleic acid. For example, IC₅₀ or K_(d) thresholdvalues of 10 μM or less, 1 μM or less, and 100 nM or less are oftenutilized. In another example, threshold values of 10 nM or less, 1 nM orless, 100 pM or less, and 10 pM or less may be utilized to identifyquadruplex interacting molecules and quadruplex forming nucleic acids.

Many assays are available for identifying compounds that have affinityfor quadruplex forming regions of DNA. In some of these assays, thebiological activity is the quadruplex nucleic acid binding to a compoundand binding is measured as a signal. In other assays, the biologicalactivity is a polymerase arresting function of a quadruplex and thedegree of arrest is measured as a decrease in a signal. In certainassays, the biological activity is transcription and transcriptionlevels can be quantified as a signal. In another assay, the biologicalactivity is cell death and the number of cells undergoing cell death isquantified. Another assay monitors proliferation rates of cancer cells.Examples of assays are fluorescence binding assays, gel mobility shiftassays (see, e.g., Jin & Pike, Mol. Endocrinol. (1996) 10:196-205),polymerase arrest assays, transcription reporter assays, cancer cellproliferation assays, and apoptosis assays (see, e.g., AmershamBiosciences (Piscataway, N.J.)), and embodiments of such assays aredescribed hereafter. Also, topoisomerase assays can be utilized todetermine whether the quadruplex interacting molecules have atopoisomerase pathway activity (see, e.g., TopoGEN, Inc. (Columbus,Ohio)).

Gel Electrophoretic Mobility Shift Assay (EMSA)

An EMSA is useful for determining whether a nucleic acid forms aquadruplex and whether a nucleotide sequence isquadruplex-destabilizing. EMSA is conducted as described previously (Jin& Pike, Mol. Endocrinol. 10: 196-205 (1996)) with minor modifications.Generally, synthetic single-stranded oligonucleotides are labeled in the5′-terminus with T4-kinase in the presence of [γ-³²P] ATP (1,000mCi/mmol, Amersham Life Science) and purified through a sephadex column.³²P-labeled oligonucleotides (˜30,000 cpm) are then incubated with orwithout various concentrations of a testing compound in 20 μl of abuffer containing 10 mM Tris pH 7.5, 100 mM KCl, 5 mM dithiothreitol,0.1 mM EDTA, 5 mM MgCl₂, 10% glycerol, 0.05% Nonedit P-40, and 0.1 mg/mlof poly(dI-dC) (Pharmacia). After incubation for 20 minutes at roomtemperature, binding reactions are loaded on a 5% polyacrylamide gel in0.25×Tris borate-EDTA buffer (0.25×TBE, 1×TBE is 89 mM Tris-borate, pH8.0, 1 mM EDTA). The gel is dried and each band is quantified using aphosphoimager.

DMS Methylation Protection Assay

Chemical footprinting assays are useful for assessing quadruplexstructure. Quadruplex structure is assessed by determining whichnucleotides in a nucleic acid are protected or unprotected from chemicalmodification as a result of being inaccessible or accessible,respectively, to the modifying reagent. A DMS methylation assay is anexample of a chemical footprinting assay. In such an assay, bands fromEMSA are isolated and subjected to DMS-induced strand cleavage. Eachband of interest is excised from an electrophoretic mobility shift geland soaked in 100 mM KCl solution (300 μl) for 6 hours at 4° C. Thesolutions are filtered (microcentrifuge) and 30,000 cpm (per reaction)of DNA solution is diluted further with 100 mM KCl in 0.1×TE to a totalvolume of 70 μl (per reaction). Following the addition of 1 μl salmonsperm DNA (0.1 g/μl), the reaction mixture is incubated with 1 μl DMSsolution (DMS:ethanol; 4:1; v:v) for a period of time. Each reaction isquenched with 18 μl of stop buffer (b-mercaptoathanol:water:NaOAc (3 M);1:6:7; v:v:v). Following ethanol precipitation (twice) and piperidinecleavage, the reactions are separated on a preparative gel (16%) andvisualized on a phosphoimager.

Polymerase Arrest Assay

An arrest assay includes a template nucleic acid, which may comprise aquadruplex forming sequence, and a primer nucleic acid which hybridizesto the template nucleic acid 5′ of the quadruplex-forming sequence. Theprimer is extended by a polymerase (e.g., Taq polymerase), whichadvances from the primer along the template nucleic acid. In this assay,a quadruplex structure can block or arrest the advance of the enzyme,leading to shorter transcription fragments. Also, the arrest assay maybe conducted at a variety of temperatures, including 45° C. and 60° C.,and at a variety of ion concentrations.

An example of the Taq polymerase stop assay is described in Han, et al.,Nucl. Acids Res. (1999) 27:537-542, which is a modification of that usedby Weitzmann, et al., J. Biol. Chem. (1996) 271:20958-20964. Briefly, areaction mixture of template DNA (50 nM), Tris.HCl (50 mM), MgCl₂ (10mM), DTT (0.5 mM), EDTA (0.1 mM), BSA (60 ng), and 5′-end-labeledquadruplex nucleic acid (˜18 nM) is heated to 90° C. for 5 minutes andallowed to cool to ambient temperature over 30 minutes. Taq Polymerase(1 μl) is added to the reaction mixture, and the reaction is maintainedat a constant temperature for 30 minutes. Following the addition of 10μl stop buffer (formamide (20 ml), 1 M NaOH (200 μl), 0.5 M EDTA (400μl), and 10 mg bromophenol blue), the reactions are separated on apreparative gel (12%) and visualized on a phosphoimager. Adeninesequencing (indicated by “A” at the top of the gel) is performed usingdouble-stranded DNA Cycle Sequencing System from Life Technologies. Thegeneral sequence for the template strands isTCCAACTATGTATAC-INSERT-TTAGCGACACGCAATTGCTATAGTGAGTCGTATTA, where“INSERT” refers to a nucleic acid sequence comprising a quadruplexforming sequence (See e.g., Table A). Bands on the gel that exhibitslower mobility are indicative of quadruplex formation.

High Throughput Polymerase Arrest Assay

A high throughput polymerase arrest assay has been developed. The assaycomprises contacting a template nucleic acid, often DNA, with a primer,which also is often DNA; contacting the primer/template complex with acompound described herein (also referred to as a “test compound”);contacting the primer/template complex with a polymerase; and separatingreaction products. The assay often includes the step of denaturing theprimer/template complex mixture and then renaturing the complex, whichoften is carried out before a test molecule is added to the system.Multiple assays often are carried out using varying concentrations of atest compound, such that an IC₅₀ value can be obtained, for example. Thereaction products often include extended primers of different lengths.Where a test compound does not significantly interact with a quadruplexstructure in the template, the primer often is extended to the end ofthe template.

Where a test compound significantly interacts with a quadruplexstructure in the template, the primer often is extended only to thequadruplex structure in the template and no further. Thus, the reactionmixture often includes at least two reaction products when a testcompound interacts with a quadruplex structure in the template, onehaving a completely extended primer and one having an incompletelyextended primer, and these two reaction products are separated. Theproducts may be separated using any convenient separation method, suchas mass spectrometry and in one embodiment, capillary electrophoresis.

The reaction products often are identified by detecting a detectablelabel linked to the primer. The detectable label may be non-covalentlylinked to the 5′ end of the primer (e.g., a biotin molecule covalentlylinked to the 5′ end of the primer which is non-covalently linked to anavidin molecule joined to a detectable label). The detectable label maybe joined to the primer at any stage of the assay, sometimes before theprimer is added to the system, after the primer is extended, or afterthe products are separated. The detectable label often is covalentlylinked to the primer using a procedure selected based upon the nature ofthe chemical groups in the detectable label.

Many methods for covalently linking detectable labels to nucleic acidsare available, such as chemically coupling an allylamine-derivatizednucleotide to a succinimidyl-ester derivative of a detectable label, andthen generating a primer using the labeled nucleotide. (See, e.g.,Nature Biotech (2000) 18:345-348 and http addressinfo.med.yale.edu/genetics/ward/tavi/n_coupling.html). A spacer (oftenbetween 5-16 carbon atoms long) sometimes is incorporated between thedetectable label and the nucleotide. Any convenient detectable label maybe utilized, including but not limited to a radioactive isotope (e.g.,¹²⁵I, ¹³¹I, ³⁵S, ³²P, ¹⁴C or ³H); a light scattering label (e.g., aspherical gold or silver label; Genicon Sciences Corporation, San Diego,Calif. and U.S. Pat. No. 6,214,560); an enzymic or protein label (e.g.,GFP or peroxidase); or another chromogenic label or dye sometimes isutilized. Often, a fluorescent label is utilized (e.g., amino-methylcoumarin (AMCA); diethyl aminomethyl coumarin (DEAC); cascade blue (CB);fluorescein isothiocyanate (FITC); Oregon green (OG); Alexa 488 (A488);rhodamine green (RGr); lanthanide chelate (e.g., europium),carboxy-rhodamine 6G (R6G); tetramethyl rhodamine (TAMRA); Texas Red(TxR); Cy3; Cy3.5; Cy5, Cy5.5 and carboxynaphtofluorescein (CNF),digoxigenin (DIG); and 2,4-dinitrophenyl (DNP)). Other fluorophores andattendant excitation and emission wavelengths are described in Anantha,et al., Biochemistry (1998) 37:2709-2714 and Qu & Chaires, MethodsEnzymol (2000) 321:353-69).

In an embodiment, a primer oligonucleotide covalently linked to afluorescent label is contacted with template DNA. The resulting complexis contacted with a test molecule and then contacted with a polymerasecapable of extending the primer. The reaction products then areseparated and detected by capillary electrophoresis. A longer primersequence was used for practicing this embodiment as compared toembodiments where the primer includes no covalently-linked fluorophoreor where capillary electrophoresis is not utilized for separation.Deoxynucleotides are added at any stage of the assay before theseparation, often when the primer is contacted with the template DNA.The template DNA/primer complex often is denatured (e.g., by increasingthe temperature of the system) and then renatured (e.g., by cooling thesystem) before a test compound is added).

The following is a specific example of the assay embodiment. A5′-fluorescent-labeled (FAM) primer (P45, 15 nM) was mixed with templateDNA (15 nM) in a Tris-HCL buffer (15 mM Tris, pH 7.5) containing 10 mMMgCl₂, 0.1 mM EDTA and 0.1 mM mixed deoxynucleotide triphosphates(dNTP's). The FAM-P45 primer (5′-6FAM-AGTCTGACTGACTGTACGTAGCTAATACGACTCACTATAGCAATT-3′) and the template DNA(5′-TCCAACTATGTATACTGGGGAGGGTGGGGAGGGTGGGGAAGGTTAGCGACACGCAATTGCTATAGTGAGTCGTATTAGCTACGTACAGTCAGTCAGACT-3′) were synthesized andHPLC purified by Applied Biosystems. The mixture was denatured at 95° C.for 5 minutes and, after cooling down to room temperature, was incubatedat 37° C. for 15 minutes.

After cooling down to room temperature, 1 mM KCl₂ and the test compound(various concentrations) were added and the mixture incubated for 15minutes at room temperature. The primer extension was performed byadding 10 mM KCl and Taq DNA Polymerase (2.5 U/reaction, Promega) andincubating at 70° C. for 30 minutes. The reaction was stopped by adding1 μl of the reaction mixture to 10 μl Hi-Di Formamide mixed and 0.25 μlLIZ120 size standard. Hi-Di Formamide and LIZ120 size standard werepurchased from Applied Biosystems. The partially extended quadruplexarrest product was between 61 or 62 bases long and the full-lengthextended product was 99 bases long. The products were separated andanalyzed using capillary electrophoresis. Capillary electrophoresis wasperformed using an ABI PRISM 3100-Avant Genetic Analyzer. The assay wasperformed using compounds described above and results are shown inTable 1. μM concentrations reported in Table 1 are concentrations atwhich 50% of the DNA was arrested in the assay (i.e., the ratio ofshorter partially extended DNA (arrested DNA) to full-length extendedDNA is 1:1).

Transcription Reporter Assay

In a transcription reporter assay, test quadruplex DNA is coupled to areporter system, such that a formation or stabilization of a quadruplexstructure can modulate a reporter signal. An example of such a system isa reporter expression system in which a polypeptide, such as luciferaseor green fluorescent protein (GFP), is expressed by a gene operablylinked to the potential quadruplex forming nucleic acid and expressionof the polypeptide can be detected. As used herein, the term “operablylinked” refers to a nucleotide sequence which is regulated by a sequencecomprising the potential quadruplex forming nucleic acid. A sequence maybe operably linked when it is on the same nucleic acid as the quadruplexDNA, or on a different nucleic acid. An exemplary luciferase reportersystem is described herein.

A luciferase promoter assay described in He, et al., Science (1998)281:1509-1512 often is utilized for the study of quadruplex formation.Specifically, a vector utilized for the assay is set forth in reference11 of the He, et al., document. In this assay, HeLa cells aretransfected using the lipofectamin 2000-based system (Invitrogen)according to the manufacturer's protocol, using 0.1 μg of pRL-TK(Renilla luciferase reporter plasmid) and 0.9 μg of thequadruplex-forming plasmid. Firefly and Renilla luciferase activitiesare assayed using the Dual Luciferase Reporter Assay System (Promega) ina 96-well plate format according to the manufacturer's protocol.

Circular Dichroism Assay

Circular dichroism (CD) is utilized to determine whether anothermolecule interacts with a quadruplex nucleic acid. CD is particularlyuseful for determining whether a PNA or PNA-peptide conjugate hybridizeswith a quadruplex nucleic acid in vitro. PNA probes are added toquadruplex DNA (5 μM each) in a buffer containing 10 mM potassiumphosphate (pH 7.2) and 10 or 250 mM KCl at 37° C. and then allowed tostand for 5 minutes at the same temperature before recording spectra. CDspectra are recorded on a Jasco J-715 spectropolarimeter equipped with athermoelectrically controlled single cell holder. CD intensity normallyis detected between 220 nm and 320 nm and comparative spectra forquadruplex DNA alone, PNA alone, and quadruplex DNA with PNA aregenerated to determine the presence or absence of an interaction (see,e.g., Datta, et al., JACS (2001) 123:9612-9619). Spectra are arranged torepresent the average of eight scans recorded at 100 nm/min.

Fluorescence Binding Assay

An example of a fluorescence binding assay is a system that includes aquadruplex nucleic acid, a signal molecule, and a test molecule. Thesignal molecule generates a fluorescent signal when bound to thequadruplex nucleic acid (e.g., N-methylmesoporphyrin IX (NMM)), and thesignal is altered when a test compound competes with the signal moleculefor binding to the quadruplex nucleic acid. An alteration in the signalwhen test molecule is present as compared to when test compound is notpresent identifies the test compound as a quadruplex interactingcompound.

50 μl of quadruplex nucleic acid or a nucleic acid not capable offorming a quadruplex is added in 96-well plate. A test compound also isadded in varying concentrations. A typical assay is carried out in 100μl of 20 mM HEPES buffer, pH 7.0, 140 mM NaCl, and 100 mM KCl. 50 μl ofthe signal molecule NMM then is added for a final concentration of 3 μM.NMM is obtained from Frontier Scientific Inc, Logan, Utah. Fluorescenceis measured at an excitation wavelength of 420 nm and an emissionwavelength of 660 nm using a FluroStar 2000 fluorometer (BMGLabtechnologies, Durham, N.C.). Fluorescence often is plotted as afunction of concentration of the test compound or quadruplex-targetednucleic acid and maximum fluorescent signals for NMM are assessed in theabsence of these molecules.

Cell Proliferation Assay

In a cancer cell proliferation assay, cell proliferation rates areassessed as a function of different concentrations of test compoundsadded to the cell culture medium. Any cancer cell type can be utilizedin the assay. In one embodiment, colon cancer cells are cultured invitro and test compounds are added to the culture medium at varyingconcentrations. A useful colon cancer cell line is colo320, which is acolon adenocarcinoma cell line deposited with the National Institutes ofHealth as accession number JCRB0225. Parameters for using such cells areavailable at the http address cellbank.nihs.gojp/cell/data/jcrbO225.htm.

Formulation of Compounds

As used herein, the term “pharmaceutically acceptable salts, esters andamides” includes but are not limited to carboxylate salts, amino acidaddition salts, esters and amides of the compounds, as well as thezwitterionic forms thereof, which are known to those skilled in the artas suitable for use with humans and animals. (See, e.g., Gerge, S. M.,et al., “Pharmaceutical Salts,” J. Pharm. Sci. (1977) 66:1-19, which isincorporated herein by reference.)

Any suitable formulation of the compounds described herein can beprepared. In cases where compounds are sufficiently basic or acidic toform stable nontoxic acid or base salts, administration of the compoundsas salts may be appropriate. Examples of pharmaceutically acceptablesalts are organic acid addition salts formed with acids that form aphysiological acceptable anion, for example, tosylate, methanesulfonate,acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate,α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts mayalso be formed, including hydrochloride, sulfate, nitrate, bicarbonate,and carbonate salts. Pharmaceutically acceptable salts are obtainedusing standard procedures well known in the art, for example by reactinga sufficiently basic compound such as an amine with a suitable acidaffording a physiologically acceptable anion. Alkali metal (e.g.,sodium, potassium or lithium) or alkaline earth metal (e.g., calcium)salts of carboxylic acids also are made.

A compound may be formulated as a pharmaceutical composition andadministered to a mammalian host in need of such treatment. In oneembodiment, the mammalian host is human. Any suitable route ofadministration may be used, including but not limited to oral,parenteral, intravenous, intramuscular, topical and subcutaneous routes.

In one embodiment, a compound is administered systemically (e.g.,orally) in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, compressed intotablets, or incorporated directly with the food of the patient's diet.For oral therapeutic administration, the active compound may be combinedwith one or more excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Such compositions and preparations should contain at least0.1% of active compound. The percentage of the compositions andpreparations may be varied and may conveniently be between about 2 toabout 60% of the weight of a given unit dosage form. The amount ofactive compound in such therapeutically useful compositions is such thatan effective dosage level will be obtained.

Tablets, troches, pills, capsules, and the like also may contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Any material used in preparing any unit dosageform is pharmaceutically acceptable and substantially non-toxic in theamounts employed. In addition, the active compound may be incorporatedinto sustained-release preparations and devices.

The active compound also may be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts may be prepared in a buffered solution, oftenphosphate buffered saline, optionally mixed with a nontoxic surfactant.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, triacetin, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms. The compound is sometimesprepared as a polymatrix-containing formulation for such administration(e.g., a liposome or microsome). Liposomes are described for example inU.S. Pat. No. 5,703,055 (Felgner, et al.) and Gregoriadis, LiposomeTechnology vols. I to III (2nd ed. 1993).

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient that are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. In all cases, the ultimate dosageform should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of theparticle size in the case of dispersions or by the use of surfactants.The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, buffers or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied inliquid form. Compounds often are administered as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid. Examples of useful dermatologicalcompositions used to deliver compounds to the skin are known (see, e.g.,Jacquet, et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No.4,992,478), Smith, et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S.Pat. No. 4,820,508).

Compounds may be formulated with a solid carrier, which include finelydivided solids such as talc, clay, microcrystalline cellulose, silica,alumina and the like. Useful liquid carriers include water, alcohols orglycols or water-alcohol/glycol blends, in which the present compoundscan be dissolved or dispersed at effective levels, optionally with theaid of non-toxic surfactants. Adjuvants such as fragrances andadditional antimicrobial agents can be added to optimize the propertiesfor a given use. The resultant liquid compositions can be applied fromabsorbent pads, used to impregnate bandages and other dressings, orsprayed onto the affected area using pump-type or aerosol sprayers.Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Generally, the concentration of the compound in a liquid compositionoften is from about 0.1 wt % to about 25 wt %, sometimes from about 0.5wt % to about 10 wt %. The concentration in a semi-solid or solidcomposition such as a gel or a powder often is about 0.1 wt % to about 5wt %, sometimes about 0.5 wt % to about 2.5 wt %. A compound compositionmay be prepared as a unit dosage form, which is prepared according toconventional techniques known in the pharmaceutical industry. In generalterms, such techniques include bringing a compound into association withpharmaceutical carrier(s) and/or excipient(s) in liquid form or finelydivided solid form, or both, and then shaping the product if required.The compound composition may be formulated into any dosage form, such astablets, capsules, gel capsules, liquid syrups, soft gels,suppositories, and enemas. The compositions also may be formulated assuspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensionsmay further contain substances which increase viscosity, including forexample, sodium carboxymethylcellulose, sorbitol, and/or dextran. Thesuspension may also contain one or more stabilizers.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

A useful compound dosage often is determined by assessing its in vitroactivity in a cell or tissue system and/or in vivo activity in an animalsystem. For example, methods for extrapolating an effective dosage inmice and other animals to humans are known to the art (see, e.g., U.S.Pat. No. 4,938,949). Such systems can be used for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population) of a compound. Thedose ratio between a toxic and therapeutic effect is the therapeuticindex and it can be expressed as the ratio ED₅₀/LD₅₀. The compounddosage often lies within a range of circulating concentrations for whichthe ED₅₀ is associated with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. For any compounds used in the methodsdescribed herein, the therapeutically effective dose can be estimatedinitially from cell culture assays. A dose sometimes is formulated toachieve a circulating plasma concentration range covering the IC₅₀(i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in in vitro assays,as such information often is used to more accurately determine usefuldoses in humans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

Another example of effective dose determination for a subject is theability to directly assay levels of “free” and “bound” compound in theserum of the test subject. Such assays may utilize antibody mimicsand/or “biosensors” generated by molecular imprinting techniques. Thecompound is used as a template, or “imprinting molecule”, to spatiallyorganize polymerizable monomers prior to their polymerization withcatalytic reagents. Subsequent removal of the imprinted molecule leavesa polymer matrix which contains a repeated “negative image” of thecompound and is able to selectively rebind the molecule under biologicalassay conditions (see, e.g., Ansell, et al., Current Opinion inBiotechnology (1996) 7:89-94 and in Shea, Trends in Polymer Science(1994) 2:166-173). Such “imprinted” affinity matrixes are amenable toligand-binding assays, whereby the immobilized monoclonal antibodycomponent is replaced by an appropriately imprinted matrix (see, e.g.,Vlatakis, et al., Nature (1993) 361:645-647). Through the use ofisotope-labeling, “free” concentration of compound can be readilymonitored and used in calculations of IC₅₀. Such “imprinted” affinitymatrixes can also be designed to include fluorescent groups whosephoton-emitting properties measurably change upon local and selectivebinding of compound. These changes can be readily assayed in real timeusing appropriate fiberoptic devices, in turn allowing the dose in atest subject to be quickly optimized based on its individual IC₅₀. Anexample of such a “biosensor” is discussed in Kriz, et al., AnalyticalChemistry (1995) 67:2142-2144.

Exemplary doses include milligram or microgram amounts of the compoundper kilogram of subject or sample weight, for example, about 1 microgramper kilogram to about 500 milligrams per kilogram, about 100 microgramsper kilogram to about 5 milligrams per kilogram, or about 1 microgramper kilogram to about 50 micrograms per kilogram. It is understood thatappropriate doses of a small molecule depend upon the potency of thesmall molecule with respect to the expression or activity to bemodulated. When one or more of these small molecules is to beadministered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid describedherein, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

The following examples are offered to illustrate but not to limit theinvention.

EXAMPLES

The following are exemplary procedures for synthesizing amidederivatives of heterocyclic substituted1,4-dihydro-4-oxo1,8-napthpyridine analogs.

Procedure

Ethyl 2,6-dichloro-5-fluoro-beta-oxo-3-pyridinepropionate (Aldrich) (2.0g, 5.9 mmol) was mixed with triethyl orthoformate (4.0 g) and aceticanhydride (2.0 g) and heated and stirred at 140° C. for 45 minutes. Thereaction was cooled and the resulting enol-ether solution (2) split intotwo equal halves.

To the first half of the reaction, 2-aminothiazole (500 mg) in ethanol(5 ml) was added and the resulting solution was then stirred at roomtemperature for 1 h. The solution was then evaporated to a residue,dioxane (10 ml) and potassium carbonate (0.25 g) added and the mixtureheated to reflux for 1 h. The resulting ethyl7-chloro-6-fluoro-1,4-dihydro-4-oxo-1-(thiazol-2-yl)-1,8-naphthyridine-3-carboxylatewas isolated by first filtering the mixture to remove the inorganicbyproducts and the evaporation of the mother liquors.

The crude thiazole ester was dissolved in acetic acid (10 ml) and 12MHCl (10 ml) and heated to 50° C. for 1.5 h. The resulting solution wascooled and partly evaporated until a solid formed and was collected.7-chloro-6-fluoro-1,4-dihydro-4-oxo-1-(thiazol-2-yl)-1,8-naphthyridine-3-carboxylicacid (5b) was used without further purification.

To the second half of the enol-ether solution, 2-aminobenzothiazole (750mg) in ethanol (5 ml) was added and the resulting solution was thenstirred at room temperature for 1 h. The solution was then evaporated toa residue, dioxane (10 ml) and potassium carbonate (0.25 g) added andthe mixture heated to reflux for 1 h. The resulting ethyl1-(benzo[d]thiazol-2-yl)-7-chloro-6-fluoro-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylatewas isolated by first filtering the mixture to remove the inorganicbyproducts and the evaporation of the mother liquors.

The crude benzothiazole ester was dissolved in acetic acid (10 ml) and12MHCl (10 ml) and heated to 50° C. for 3 h. The resulting solution wascooled and evaporated until a solid formed and was collected.7-chloro-6-fluoro-1,4-dihydro-4-oxo-1-(thiazol-2-yl)-1,8-naphthyridine-3-carboxylicacid (5a) was used without further purification.

Products were then reacted with amines using the following generalprocedure:

To a series of solutions of the fluoroacid (0.5 mmol) in NMP (3.6 mL)was added the amines NHR₁R₂ (0.5-2.0 mmol) at room temperature. Thevessel were sealed and heated on a 90° C. hotplate with constantstirring for 1-2 hours until the reactions were determined to becomplete by HPLC/MS analysis. The reaction mixtures were allowed to coolto room temperature and water was added (20 mL). The resultingprecipitates were collected by vacuum filtration and dried under vacuum.In cases where 1.0 equivalent of amine was used, the resulting reactionmixtures were used in the next step “as is”. The resulting solids orsolutions were treated with HBTU (2.5 eq.) and DIEA in 3.6 mL NMP andallowed to stir for 30 minutes at room temperature under an inertatmosphere. These solutions were added to a series of amines NHR₃R₄ (2.5equivalents) in a 96 well format (Whatman Uniplate, 2 mL) and allowed toreact for 2 hours. Methanol was then added (50-100 μL) and the plate wasfiltered (Whatman Unifilter Polypropylene). The resulting liquids weredirectly chromatographed on reverse HPLC (Waters Xterra 19×50 mm) withmass directed collection (Micromass ZQ, Waters FCII). The fractions wereanalyzed for purity (MS TIC, UV) and dried by vacuum evaporation(Savant), with an average yield of 5-10 mg). Structures, molecularweights, and formulas for final products are set forth in Table 1. TABLE1 mol Formula weight ID Structure Structure Structure 1.

C30H37FN8O2S 592.7442 2.

C26H27FN6O3S 522.6059 3.

C27H30FN7O2S 535.6483 4.

C28H32FN7O3S 565.6747 5.

C27H29FN6O3S 536.633 6.

C29H34FN7O2S 563.7024 7.

C28H32FN7O2S 549.6753 8.

C24H30FN7O2S 499.6148 9.

C24H32FN7O2S 501.6307 10.

C23H30FN7O2S 487.6037 11.

C25H33FN8O2S 528.6566 12.

C23H27FN6O3S 486.5724 13.

C25H32FN7O2S 513.6419

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative, and are not to be takenas limitations upon the scope of the invention. Various changes andmodifications to the disclosed embodiments will be apparent to thoseskilled in the art. Such changes and modifications, including withoutlimitation those relating to the chemical structures, substituents,derivatives, intermediates, syntheses, formulations and/or methods ofuse of the invention, may be made without departing from the spirit andscope thereof. U.S. patents and publications referenced herein areincorporated by reference.

1-27. (canceled)
 28. A method for identifying a compound that interactswith a quadruplex-forming region of DNA, comprising a) contacting anucleic acid capable of forming a quadruplex with a primer comprising alabel to form a complex; b) contacting said complex with one or moretest compounds and a polymerase to form a reaction mixture, and c)separating said reaction mixture by capillary electrophoresis to obtainone or more reaction products; and d) determining the extent of primerextension in said one or more reaction products.
 29. The method of claim28, further comprising the step of determining the binding affinity ofsaid one or more test compounds for said nucleic acid.
 30. The method ofclaim 28, wherein said label is a fluorescent label. 31-42. (canceled)43. The method of claim 28, wherein the quadruplex-forming DNA is froman oncogene DNA sequence.
 44. The method of claim 43, wherein theoncogene is the MYC, HIF, VEGF, ABL, TGF, PDGFA, MYB, SPARC, HUMTEL,HER, VAV, RET, H-RAS, EGF, SRC, BCL1, or BCL2 oncogene.
 45. The methodof claim 28, wherein the quadruplex forming DNA is from a viral DNAsequence.
 46. The method of claim 45, wherein the virus is HIV.
 47. Themethod of claim 28, wherein the primer is covalently linked to thelabel.
 48. The method of claim 28, wherein the label is a radioactiveisotope, an enzyme, a protein, or a chromagenic label.
 49. The method ofclaim 28, wherein the polymerase is Taq polymerase.
 50. The method ofclaim 28, wherein the reaction mixture further comprises an ion.
 51. Themethod of claim 50, wherein the ion is potassium.
 52. The method ofclaim 28, further comprising the step of denaturing the primer/templatecomplex mixture and then renaturing the complex prior to adding the testcompound.
 53. A method for treating a cell proliferative disorder in asubject or system, comprising administering to said subject or system,an effective amount of the compound of formula (1) or a pharmaceuticalcomposition thereof, thereby treating said cell-proliferative disorder,said compound having formula (1)

and pharmaceutically acceptable salts thereof; wherein: W is NR¹R² orNR¹—(CR¹ ₂)_(n)—NR³R⁴; Z is OR₂, NH₂, NR¹R² or NR¹—(CR¹ ₂)_(n)—NR³R⁴;wherein in NR¹R² and NR³R⁴, R¹ and R² together with N and R³ and R⁴together with N may form an optionally substituted 5-6 membered ringcontaining N, O, or S; A is H, halo or NR¹ ₂; R¹ and R³ areindependently H or a C₁₋₆ alkyl; R² is a C₁₋₁₀ alkyl or C₂₋₁₀ alkenyloptionally containing one or more non-adjacent heteroatoms selected fromN, O, and S, and optionally substituted with a C₃₋₆ cycloalkyl, aryl, ora 5-14 membered heterocyclic ring containing N, O, or S; or R² is anaryl, heteroaryl, or an optionally substituted 5-14 memberedheterocyclic ring containing N, O, or S; R⁴ is H or a C₁₋₁₀ alkyl orC₂₋₁₀ alkenyl optionally containing one or more non-adjacent heteroatomsselected from N, O, and S, and optionally substituted with a carbocyclicor a 5-6 membered heterocyclic ring; m is 1-2; n is 1-6; Y is selectedfrom the group consisting of

where R⁶ is a substituent at any position on the ring or fused ring; andis H, OR¹, C₁₋₆ alkyl, C₂₋₆ alkenyl, each optionally substituted byhalo, C═O or one or more heteroatoms; or two adjacent R⁶ is linked toobtain a 5-6 membered substituted or unsubstituted carbocyclic orheterocyclic ring, optionally fused to an additional substituted orunsubstituted carbocyclic or heterocyclic ring; Q is CH or N; and X isO, NH, or S.
 54. The method of claim 53, wherein said cell proliferativedisorder is cancer.
 55. The method of claim 53, wherein cellproliferation is reduced, or cell death is induced.
 56. The method ofclaim 53, wherein said subject is human or an animal; and said system isa cell or tissue.
 57. The method of claim 53, wherein said compound isselected from the group consisting of: